Activatable polymer nanoagonist for second near-infrared photothermal immunotherapy of cancer

Immunotherapy, while revolutionary, faces limitations such as limited response rates and adverse effects. Nanomedicine offers potential solutions by enhancing drug-immune cell interactions and enabling combination therapies. Smart immunotherapeutic nanoagents with controlled activation are crucial for clinical translation. Current approaches often rely on endogenous biomarkers (pH, ROS, enzymes), which limit bioavailability and selectivity. External stimuli, like near-infrared (NIR) light, offer better spatiotemporal and dosage control. NIR-II light (1000-1300 nm) possesses superior tissue penetration and reduced phototoxicity compared to NIR-I. Semiconducting polymer nanoparticles (SPNs) are promising NIR-II nanoagents due to their photothermal performance and biocompatibility. This study aims to synthesize and evaluate an activatable polymer nanoagonist (APNA) for NIR-II light-regulated photothermal immunotherapy, addressing the limitations of existing approaches.

Several studies have explored activatable immunotherapeutic nanoagents responsive to various stimuli. For example, human interleukin-15 super-agonist complexes with disulfide cross-linkers have been engineered for activatable release upon T-cell receptor activation. Another example involved pH-responsive conjugation of programmed cell death protein 1 antibody to magnetic nanoclusters for selective activation in acidic tumor microenvironments. NIR-I light-responsive nanoagents have been developed to convert photons into regulatory signals, such as singlet oxygen or high-energy emission to liberate immunostimulants. However, the use of NIR-II light for spatiotemporal photoregulation of immunotherapy using SPNs remains largely unexplored.

The researchers synthesized APNA by covalently conjugating Resiquimod (R848), a TLR7/8 agonist, to a NIR-II absorbing semiconducting polymer backbone (pBODO) via a thermolabile linker (VA-044). The synthesis involved Stille polycondensation of three monomers to produce PBODO-Br, followed by azide substitution to yield pBODO-N3. VA-044 was modified with 7-iodoheptanoic acid and then conjugated to R848. A bifunctional PEG was used to link the modified VA-044-R848 conjugate to pBODO-N3 via click chemistry. The resulting amphiphilic polymer self-assembled into nanoparticles (APNA). A control nanoparticle (APNC) lacking the pro-agonist was also prepared. In vitro characterization included absorption and fluorescence spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and zeta potential measurements. Photothermal properties were assessed by measuring temperature changes upon NIR-II (1064 nm) irradiation, and photothermal conversion efficiency was calculated. HPLC was used to evaluate photothermal activation of the pro-agonist. In vitro studies included cellular uptake in 4T1 cancer cells and BMDCs, cytotoxicity assays, and flow cytometry analysis of DC maturation (CD80/CD86 expression). In vivo studies utilized 4T1 tumor-bearing mice. NIR fluorescence imaging tracked nanoparticle accumulation in tumors. Treatment groups included PBS, APNC with NIR-II irradiation, APNA without irradiation, and APNA with irradiation. Tumor growth, survival, lung and liver metastasis were monitored. Flow cytometry analyzed DC maturation in tumor-draining lymph nodes and immune cell infiltration (CD8+ T cells, CD4+ T cells) in primary and distant tumors. ELISA measured cytokine levels (IL-6, IL-12p70, IFN-γ, TNF-α). Immunofluorescence staining assessed the expression of caspase-3, HMGB1, CD80, and CD86 at different tumor depths to investigate the mechanism of immune activation.

APNA exhibited strong NIR-II absorption and high photothermal conversion efficiency (84.4%). Photothermal irradiation triggered the release of the R848 agonist from APNA in a temperature-dependent manner. APNA showed effective cellular uptake in both 4T1 cancer cells and BMDCs, primarily localizing in lysosomes. NIR-II irradiation significantly enhanced the cytotoxicity of APNA to 4T1 cells. Photothermally activated APNA stimulated DC maturation, as indicated by increased CD80 and CD86 expression, significantly higher than APNC or free R848. In vivo, APNA preferentially accumulated in tumors. APNA-mediated photothermal immunotherapy nearly eradicated primary tumors and significantly inhibited the growth of distant tumors, along with reducing lung and liver metastasis. This therapy induced superior DC maturation in tumor-draining lymph nodes and increased levels of proinflammatory cytokines. Importantly, APNA-mediated photothermal immunotherapy resulted in significantly higher infiltration of CD8+ T cells and CD4+ T cells into both primary and distant tumors compared to other treatments. Mechanistic studies revealed that while both photothermal therapy (PTT) and photothermal immunotherapy induced similar levels of apoptosis and immunogenic cell death, photothermal immunotherapy significantly enhanced DC maturation, especially at the same temperature. This enhancement was attributed to the in situ release of the TLR agonist from APNA.

The study successfully demonstrated the efficacy of APNA-mediated NIR-II photothermal immunotherapy in combating deep solid tumors and metastasis. The activatable design of APNA allows for precise spatiotemporal control of immune activation, minimizing off-target effects. The use of SPNs as the NIR-II photothermal transducer provides excellent biocompatibility and avoids potential toxicity associated with inorganic materials. The controlled release of the TLR7/8 agonist overcomes the limitations of systemic administration of R848 alone. The findings highlight the synergistic effect of combining photothermal ablation, immunogenic cell death induction, and in situ immune activation. The mechanistic studies shed light on the process of intratumoral immune activation at different depths and temperatures. The superior antitumor efficacy was dependent on T cells, highlighting the importance of the immune response in this combined approach.

This research successfully developed a NIR-II photothermally activatable polymeric pro-nanoagonist for combinational photothermal immunotherapy, achieving significant antitumor efficacy and metastasis inhibition. This approach offers a promising strategy for remote and precise control of immune activation in cancer therapy. Future research could explore the application of this strategy with other immunostimulants or checkpoint inhibitors and investigate potential clinical translations.

The study was conducted using a murine model, and further investigations are necessary to confirm the findings in human clinical trials. While the authors carefully addressed the potential side effects by using an activatable design, long-term toxicity studies are still needed to ensure the safety of this approach. The precise mechanism of action of APNA may require further investigation at a molecular level. The study focused on 4T1 breast cancer cells, which may limit the generalizability of these findings to other cancer types.